T lymphocytes compose a major part of the body's immune defenses against bacterial, viral and protozoal infection, and have been implicated in the rejection of cancerous cells. Numerous autoimmune syndromes have also been linked to antigen-specific T cell attack on various parts of the body. It is therefore of great interest to be able to track antigen-specific T cells during the course of these diseases. It would also be of great therapeutic benefit if T cells specific for a particular antigen could be enriched and then reintroduced in a disease situation, or selectively depleted in the case of an autoimmune disorder.
Current technology for isolating or otherwise identifying antigen-specific T cells is very crude. While the T cell antigen receptors (TCR) are structurally and genetically similar to antibodies, their ligands are complex, consisting of a peptide embedded in a molecule of the major histocompatibility complex. MHC proteins come in two basic types, class I and class II, each of which subserves a particular type of T cell. Class I molecules interact with CD8.sup.+ T cells, whereas class II molecules interact with CD4.sup.+ T cells. The two types of MHC molecules differ in the length of peptide which they are able to present. The class I peptide binding pocket is blocked at either end, thereby imposing severe restrictions on the sizes of peptides it can accommodate (8-10 residues), with longer peptides bulging out in the middle. The class II binding groove allows peptides to protrude from the ends, and consequently much longer peptides, (8-30 residues) can bind.
Production of monoclonal antibody reagents specific for various T cell variable region epitopes has been difficult. Further, labeling cells with these reagents is of limited utility when one is interested in characterizing T cells having a particular antigenic specificity. Monoclonal antibodies are usually directed to the TCR variable region, including relatively conserved sequences, not to the actual TCR binding site. Monoclonal antibodies may detect T cells having the same variable region but different antigenic specificity, while failing to detect T cells with the same antigenic specificity but a different variable region.
Previous attempts to use MHC protein-peptide complexes to label specific T cells have been unsuccessful, primarily due to the short half-life of the peptide-MHC-T cell receptor ternary complex. At room temperature, the half-life of the ternary complex is only 12 to 25 seconds. Because of the crucial role that the T-cell plays as a major component of the immune system, it remains of great importance to be able to understand how T-cells are selected, activated or tolerized. Specific tracking and the ability to physically purify or ablate antigen specific T cells is of particular interest.
Relevant Literature
The molecular structure of a class I major histocompatibility antigen is described in Bjorkman et al. (1987) Nature 329:506-512. The structure of a class II MHC antigen is described in J. Brown et al. (1993) Nature 364:33-39. The low affinity of the ternary complex formed between T cell receptor, MHC antigen and antigenic peptide is described in K. Matsui et al. (1991) Science 254:1788-1791. While affinities of T cell receptors range from 10.sup.-4 to 10.sup.-7 M, the t1/2, which governs the complex stability, is very similar between high and low affinity receptors, ranging from 12 to 25 seconds at room temperature.
Refolding and crystallization of class I MHC--peptide complexes is described in Garboczi et al. (1992) P.N.A.S. 89:3429-3433. Formation of functional class II complexes is described in Altman et al. (1993) P.N.A.S. 90: 10330-10334.
The substrate specificity of the E. coli BirA enzyme is described in P. Schatz (1993) Bio/Technology 11:1138-1143.
Various methods for analyzing peptide binding to T cell receptor and to major histocompatibility complex antigens have been explored. Fridkis-Hareli et al. (1994) P.N.A.S. 91:4872-4876 discuss the binding of peptides to class It MHC molecules on living cells. A europium fluoroimmunoassay has also been used to measure binding of antigen to class II MHC proteins, Tompkins et al. (1993) J. lmmunol. Meth. 163:209-216. Photoaffinity labeling of the T cell receptor is discussed in Romero et al. (1993) J. Immunol. 150:3825-3831.